Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae.

Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-ß-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications.

Biocatalytic characterization of Hericium erinaceus laccase isoenzymes for the oxidation of lignin derivative substrates.

Mushroom laccases are biocatalysts that oxidize various substrates. To identify a novel enzyme involved in lignin valorization, we isolated and characterized laccase isoenzymes from the mushroom Hericium erinaceus. The laccase cDNAs (Lac1a and Lac1b) cloned from the mushroom mycelia consisted of 1536 bp and each encoded a protein with 511 amino acids, containing a 21-amino-acid signal peptide. Comparative phylogenetic analysis revealed high homology between the deduced amino acid sequences of Lac1a and Lac1b and those from basidiomycetous fungi. In the Pichia pastoris expression system, high extracellular production of Lac1a, a glycoprotein, was achieved, whereas Lac1b was not expressed as a secreted protein because of hyper-glycosylation. Biochemical characterization of the purified recombinant Lac1a (rLac1a) protein revealed its oxidizing efficacy toward 14 aromatic substrates. The highly substrate-specific rLac1a showed catalytic efficiencies of 877 s-1 mM-1, 829 s-1 mM-1, 520 s-1 mM-1, and 467 s-1 mM-1 toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroquinone, guaiacol, and 2,6-dimethylphenol, respectively. Moreover, rLac1a showed approximately 10 % higher activity in non-ionic detergents and >50 % higher residual activity in various organic solvents. These results indicate that rLac1a is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products.